Roles of chlorophyllin in cell proliferation and the expression of apoptotic and cell cycle genes in HB4a non-tumor breast cells.

Chlorophyllin (Chl) has attracted interest in the scientific community due to its chemopreventive and antimutagenic properties. However, the molecular mechanisms of action of Chl remain unclear. This study assesses the effects on cell proliferation and the expression of genes involved in apoptosis, and the cell cycle in HB4a cells treated with Chl. Chl was cytotoxic and induced apoptosis to HB4a cells at 400 µg/mL. Analysis of gene expression showed that there was a decrease in the mRNA level of BIRC5 and CCNA2 genes involved in apoptosis and cell cycle progression, respectively. The proapoptotic gene BAX and the antiapoptotic genes BCL-2 and BCL-XL were upregulated. The cytotoxicity of Chl has been attributed to increases in the expression of BAX and decreases in the expression of genes involved in the cell cycle, but increases in the expression of anti-apoptotic genes suggests a mechanism for protection from cell death induced by Chl. This study provides important information about mechanisms that protect against or trigger damaging processes in non-tumor cells.

Cytotoxicity, genotoxicity and mechanism of action (via gene expression analysis) of the indole alkaloid aspidospermine (antiparasitic) extracted from Aspidosperma polyneuron in HepG2 cells.

Aspidospermine is an indole alkaloid with biological properties associated with combating parasites included in the genera Plasmodium, Leishmania and Trypanossoma. The present study evaluated the cytotoxicity (resazurin test), genotoxicity (comet assay) and mechanism of action (gene expression analysis via qRT-PCR) of this alkaloid in human HepG2 cells. The results demonstrated that treatment with aspidospermine was both cytotoxic (starting at 75 µM) and genotoxic (starting at 50 µM). There was no significant modulation of the expression of the following genes: GSTP1 and GPX1 (xenobiotic metabolism); CAT (oxidative stress); TP53 and CCNA2 (cell cycle); HSPA5, ERN1, EIF2AK3 and TRAF2 (endoplasmic reticulum stress); CASP8, CASP9, CASP3, CASP7, BCL-2, BCL-XL BAX and BAX (apoptosis); and PCBP4, ERCC4, OGG1, RAD21 and MLH1 (DNA repair). At a concentration of 50 µM (non-cytotoxic, but genotoxic), there was a significant increase in the expression of CYP1A1 (xenobiotic metabolism) and APC (cell cycle), and at a concentration of 100 µM, a significant increase in the expression of CYP1A1 (xenobiotic metabolism), GADD153 (endoplasmic reticulum stress) and SOD (oxidative stress) was detected, with repression of the expression of GR (xenobiotic metabolism and oxidative stress). The results of treatment with aspidospermine at a 100 µM concentration (the dose indicated in the literature to achieve 89 % reduction of the growth of L. amazonensis) suggest that increased oxidative stress and an unfolded protein response (UPR) occurred in HepG2 cells. For the therapeutic use of aspidospermine (antiparasitic), chemical alteration of the molecule to achieve a lower cytotoxicity/genotoxicity in host cells is recommended.

Changes in gene expression in PBMCs profiles of PPARα target genes in obese and non-obese individuals during fasting.

BACKGROUND: The prevalence of obesity has risen dramatically and the World Health Organization estimates that 700 million people will be obese worldwide by 2015. Approximately, 50% of the Brazilian population above 20 years of age is overweight, and 16% is obese. AIM: This study aimed to evaluate the differences in the expression of PPARα target genes in human peripheral blood mononuclear cells (PBMCs) and free fatty acids (FFA) in obese and non-obese individuals after 24 h of fasting. We first presented evidence that Brazilian people exhibit expression changes in PPARα target genes in PBMCs under fasting conditions. METHODS: Q-PCR was utilized to assess the mRNA expression levels of target genes. RESULTS: In both groups, the FFA concentrations increased significantly after 24 h of fasting. The basal FFA mean concentration was two-fold higher in the obese group compared with the non-obese group. After fasting, all genes evaluated in this study showed increased expression levels compared with basal expression in both groups. CONCLUSION: However, our results reveal no differences in gene expression between the obese and non-obese, more studies are necessary to precisely delineate the associated mechanisms, particularly those that include groups with different degrees of obesity and patients with diabetes mellitus type 2 because the expression of the main genes that are involved in ß-oxidation and glucose level maintenance are affected by these factors.

Cytotoxic, biochemical and genotoxic effects of biodiesel produced by different routes on ZFL cell line.

Transesterification has proved to be the best option for obtaining biodiesel and, depending on the type of alcohol used in the reaction, the type of biodiesel may be methyl ester or ethyl ester. Leaking biodiesel can reach water bodies, contaminating aquatic organisms, particularly fish. The objective of this study was to determine whether the soluble fraction of biodiesel (Bd), produced by both the ethylic (BdEt) and methylic (BdMt) routes, can cause cytotoxic, biochemical and genotoxic alterations in the hepatocyte cell line of Danio rerio (ZFL). The metabolic activity of the cell was quantified by the MTT reduction method, while genotoxic damage was analyzed by the comet assay with the addition of specific endonucleases. The production of reactive oxygen species (ROS) and antioxidant/biotransformation enzymes activity also were determined. The results indicate that both Bd increased ROS production, glutathione S-transferase activity and the occurrence of DNA damage. BdMt showed higher cytotoxicity than BdEt, and also caused oxidative damage to the DNA. In general, both Bd appear to be stressors for the cells, causing cytotoxic, biochemical and genetic alterations in ZFL cells, but the type and intensity of the changes found appear to be dependent on the biodiesel production route.

Modulação do efeito mutagênico do benzo[a]pireno e bleomicina por extratos de isoflavonas em células de hepatoma de roedor / Modulation of the mutagenic effect of benzo[a]pyrene and bleomycin by isoflavone extracts in a rat hepatoma cell line

Estudos epidemiológicos mostram que a ingestão de alimentos ricos em isoflavonas (fitoestrógenos), como a soja, confere proteção contra vários tipos de câncer, o que aumenta o interesse científico e popular sobre esses compostos. No presente estudo, os fitoestrógenos de extrato de soja foram testados quanto aos efeitos genotóxicos e modulador de benzo [a] pireno e bleomicina. Dois fitoestrogênios foram avaliados in vitro, o fitoestrógenos “A” foi fornecido pela Embrapa-Soja, Londrina - PR, e o fitoestrógenos “B” foi comprado em uma farmácia de manipulação local. Os métodos utilizados foram o teste do Cometa (genotoxicidade e antigenotoxicidade) e teste do Micronúcleo com Bloqueio Citocinese mutagenicidade) em células de hepatoma de rato (HTC celulares). As isoflavonas foram testadas em três concentrações pré-estabelecidas pelo ensaio de citotoxidade MTT. Ambos os extratos de isoflavonas não mostraram efeitos genotóxicos no ensaio do cometa, mas mostraram indução de micronúcleo. Na avaliação dos fitoestrogênios para um efeito modulador, ambos os extratos fitoestrogênios mostraram efeito antigenotóxico no ensaio do cometa.

Epidemiologic studies show that the intake of foods rich in isoflavones (phytoestrogens), such as soybeans, confers protection against various types of cancer, what increases the scientific and popular interest on these compounds. In the present study, phytoestrogens extracts from soybeans were tested for genotoxic potential and modulatory effects on benzo[a]pyrene and bleomycin. Two phytoestrogens were evaluated in vitro, phytoestrogen “A” was supplied by EMBRAPA-Soja, Londrina – PR, and phytoestrogen “B” was purchased in a local drug store. The methods used were the comet assay (genotoxicity and antigenotoxicity) and micronucleus test with cytokinesis block (mutagenicity) in rat hepatoma cells (HTC cell). The isoflavones were tested at three concentrations pre-established by the MTT cytotoxicity assay. Both isoflavone extracts showed no genotoxic effects in the comet assay, but showed induction of micronucleus. In the evaluation of the phytoestrogens for a modulatory effect, both phytoestrogens extracts showed antigenotoxicity in the comet assay.

Evaluation of extracts from Coccoloba mollis using the Salmonella/microsome system and in vivo tests.

The common everyday use of medicinal plants is an ancient, and still very widespread practice, whereby the need for studies on their possible toxicity and mutagenic properties. The species Coccoloba mollis has been much used in phytotherapy, mainly in cases involving loss of memory and stress. In order to investigate its genotoxic and mutagenic potential, ethanolic extracts from the leaves and roots underwent Salmonella/microsome assaying (TA98 and TA100 strains, with and without exogenous metabolism - S9), besides comet and micronucleus tests in vivo.There was no significant increase in the number of revertants/plate of Salmonella strains in any of the analyzed root-extract concentrations, although the extract itself was extremely toxic to the Salmonella TA98 strain in the tests carried out with S9 (doses varying from 0.005 to 0.5 µg/plate). On the other hand, the leaf-extract induced mutations in the TA98 strain in the absence of S9 in the highest concentration evaluated, although at very low mutagenic potency (0.004 rev/ µg). Furthermore, there was no statistically significant increase in the number of comets and micronuclei, in treatments involving Swiss mice. It was obvious that extracts of Coccoloba mollis, under the described experimental conditions, are not mutagenic.

Evaluation of extracts from Coccoloba mollis using the Salmonella/microsome system and in vivo tests

The common everyday use of medicinal plants is an ancient, and still very widespread practice, whereby the need for studies on their possible toxicity and mutagenic properties. The species Coccoloba mollis has been much used in phytotherapy, mainly in cases involving loss of memory and stress. In order to investigate its genotoxic and mutagenic potential, ethanolic extracts from the leaves and roots underwent Salmonella/microsome assaying (TA98 and TA100 strains, with and without exogenous metabolism - S9), besides comet and micronucleus tests in vivo.There was no significant increase in the number of revertants/plate of Salmonella strains in any of the analyzed root-extract concentrations, although the extract itself was extremely toxic to the Salmonella TA98 strain in the tests carried out with S9 (doses varying from 0.005 to 0.5 µg/plate). On the other hand, the leaf-extract induced mutations in the TA98 strain in the absence of S9 in the highest concentration evaluated, although at very low mutagenic potency (0.004 rev/µg). Furthermore, there was no statistically significant increase in the number of comets and micronuclei, in treatments involving Swiss mice. It was obvious that extracts of Coccoloba mollis, under the described experimental conditions, are not mutagenic.

The azo dyes Disperse Red 1 and Disperse Orange 1 increase the micronuclei frequencies in human lymphocytes and in HepG2 cells.

The use of azo dyes by different industries can cause direct and/or indirect effects on human and environmental health due to the discharge of industrial effluents that contain these toxic compounds. Several studies have demonstrated the genotoxic effects of various azo dyes, but information on the DNA damage caused by Disperse Red 1 and Disperse Orange 1 is unavailable, although these dyes are used in dyeing processes in many countries. The aim of the present study was to evaluate the mutagenic activity of Disperse Red 1 and Disperse Orange 1 using the micronucleus (MN) assay in human lymphocytes and in HepG2 cells. In the lymphocyte assay, it was found that the number of MN induced by the lowest concentration of each dye (0.2 microg/mL) was similar to that of the negative control. At the other concentrations, a dose response MN formation was observed up to 1.0 microg/mL. At higher dose levels, the number of MN decreased. For the HepG2 cells the results were similar. With both dyes a dose dependent increase in the frequency of MN was detected. However for the HepG2, the threshold for this increase was 2.0 microg/mL, while at higher doses a reduction in the MN number was observed. The proliferation index was also calculated in order to evaluate acute toxicity during the test. No differences were detected between the different concentrations tested and the negative control.